gas chromatography–mass spectrometry determination of metabolites of conjugated...

Journal of Chromatography B, 820 (2005) 15–22

Gas chromatography–mass spectrometry determinationof metabolites of conjugatedcis-9,trans-11,

cis-15 18:3 fatty acid

Frederic Destaillatsa, Jean-Louis Sebediob, Olivier Berdeauxb,Pierre Juanedab, Paul Angersa,∗

a Department of Food Science and Nutrition, and Dairy Research Center (STELA), Universit´e Laval,Quebec, QC, Canada, G1K 7P4

b INRA, Unite de Nutrition Lipidique, 17 rue Sully, Dijon, France

Received 20 January 2005; accepted 28 February 2005

Abstract

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Structural determination of polyunsaturated fatty acids by gas chromatography–mass spectrometry (GC–MS) requires cuse of nitrogen containing derivatives such as picolinyl esters, 4,4-dimethyloxazoline or pyrrolidides derivatives. The deri

s required in most cases to obtain low energy fragmentation that allows accurate location of the double bonds. In thork, the following metabolites of rumelenic (cis-9,trans-11,cis-15 18:3) acid, from rat livers, were identified:cis-8,cis-11,trans-13,cis-7 20:4,cis-5,cis-8,cis-11,trans-13,cis-17 20:5,cis-7,cis-10,cis-13,trans-15,cis-19 22:5, andcis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6cids by GC–MS as their 4,4-dimethyloxazoline and methyl esters derivatives. Specific fragmentation of the methyl est

ives revealed some similarity with their corresponding DMOX derivatives. Indeed, intense ion fragments atm/z= M+ − 69, correponding to a cleavage at the center of a bis-methylene interrupted double bond system were observed for all identifieites. Moreover, intense ion fragments atm/z= M+ − 136, corresponding to allylic cleavage of then-12 double bonds were oerved for the C20:5, C22:5, C22:6 acid metabolites. For the long chain polyunsaturated fatty acids from the rumelenic me showed that single methyl esters derivatives might be used for both usual quantification by GC–FID and identificC–MS.2005 Elsevier B.V. All rights reserved.

eywords: Conjugated fatty acid metabolites; Gas chromatography–mass spectrometry; Long chain polyunsaturated fatty acid; Rat liver

. Introduction

Gas chromatography–mass spectrometry (GC–MS) islassically used for structural determination of unsaturatedatty acids[1]. Methyl esters prepared at room temperaturey alkali-catalyzed transesterification are currently used for

atty acid profile determination by GC–FID[2]. These deriva-ives rarely provide characteristic ion fragments that allowccurate location of the ethylenic double bonds[1]. In order

∗ Corresponding author. Tel.: +1 418 656 2843; fax: +1 418 656 3353.E-mail address:[email protected] (P. Angers).

to obtain low ionization potential, the carboxyl group mbe derivatized with a reagent containing a nitrogen atom[1].In this case, the nitrogen group carries the charge andble bond migration and ionization are subsequently mmized[1]. Picolinyl esters[3,4] and 4,4-dimethyloxazoline(DMOX) [5] are extensively used, and their GC–MS prerties have been reviewed in details by Christie[1]. DMOXderivatives exhibit good chromatographic properties[1,3]; in-deed, only slight differences in the temperature programare necessary to obtain similar retention times as forcorresponding fatty acid methyl esters (FAME). Howedrastic thermal conditions (150–190◦C) and long reactio

570-0232/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.jchromb.2005.02.021

16 F. Destaillats et al. / J. Chromatogr. B 820 (2005) 15–22

time (6–12 h) are necessary to obtain sufficient derivatizationyield, resulting in partial degradation of sensitive samplessuch as long chain polyunsaturated fatty acids (PUFA)[1].Picolinyl esters can be prepared by transesterification suchas methyl esters at moderate temperature[4], but their anal-ysis requires column temperatures about 50◦C higher thanfor methyl esters[1]. Using appropriate stationary phases,the resolution problem is greatly lessened but resolutionof PUFA tends to be difficult particularly in complex mix-tures.

In the present study, structural identification of somemetabolites in the rat of rumelenic (cis-9,trans-11,cis-1518:3) acid, a minor component of milk fat, was achievedusing both DMOX and FAME. Mass spectra of both deriva-tives were compared and the results showed that FAMEcan efficiently be used for GC–MS characterization of thesepartially conjugated long chain PUFA in complex sam-ples.

2. Materials and methods

2.1. Samples

Liver lipid samples from rats fed a mixture of conjugatedcis-9,trans-11,cis-15 18:3 andcis-9,trans-13,cis-15 18:3 iso-m iaI viouss

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3. Results and discussion

Rumelenic (cis-9,trans-11,cis-15 18:3) acid, a minor com-ponent of ruminant fats[7–9], is an intermediate of the “bio-hydrogenation” process of�-linolenic (cis-9,cis-12,cis-1518:3) acid in the rumen. Metabolism of conjugated dienoicFA has been extensively studied over the past years[10–13].Indeed, by analogy, we have studied the metabolism of therumelenic acid[5] in the rat resulting in the proposed bio-chemical pathway (Fig. 1).

The octadecatetraenoic intermediate has not been de-tected but the elongation product, thecis-8,cis-11,trans-13,cis-17 20:4 acid, has been identified from total liverlipid. The spectrum of its DMOX derivative is shown inFig. 2A. The molecular ion atm/z= 357 and the loss of theterminal methyl group (m/z= 342) confirmed the eicosate-traenoic acid structure. Ion fragments atm/z= 328 and 302allow the location of an ethylenic double bond in posi-tion �17, which is corroborated by the intense ion frag-ment at m/z= 288. Such characteristic fragment is typ-ical for bis-methylene interrupted double bond systems[1,14,15].

Ions fragments atm/z= 274, 260, 248, 234, and 222 al-low the location of the 11,13-conjugated system, while thedouble bond at position 8 is confirmed either by ion frag-ments atm/z= 182 and 194 or allylic cleavage fragments atm thei f ar trum

F (9,trans-11,cis-15 18:3) acid (adapted from[5]).

ers of�-linolenic acid (CLNA) kindly donated by Naturnc. (Sherbrooke, PQ, Canada) were obtained from a pretudy[5].

.2. Preparation of fatty acid derivatives (FAME)

Methylation ofO-acetylated fatty acids was carriedith sodium methoxide in methanol[2]. DMOX derivativesf FA were prepared using modified literature proced

3,6] by heating total liver lipids (10 mg) directly withmino-2-methyl-1-propanol (500�L) under nitrogen atmophere at 150◦C overnight.

.3. GC–MS analysis of FAME and DMOX derivatives

FAME and DMOX derivatives were analyzedC–MS (Hewlett-Packard model 6890 Series II gas catograph attached to an Agilent model 5973N

ective quadrupole mass detector; Palo Alto, CA)er an ionization voltage of 70 eV at 230◦C, and conected to a computer with a Hewlett-Packard Chem

ion. The injector (splitless mode) and the interface teratures were maintained at 250◦C, while He was uses carrier gas under constant flow (2.4 mL min−1). GCeparation was performed on a BPX-70 capillarymn (SGE, Melbourne, Australia; 50 m, 0.32 mm i.25�m film thickness). Temperature programming monsisted of 60◦C isothermal for 1 min, increased70◦C at 20◦C min−1, and held isothermal for 40 min70◦C.

/z= 222 and 168. The key diagnostic ion for this FA isntense ion fragment atm/z= 288 representing the loss oadical fragment of 69 atomic mass unit (amu). The spec

ig. 1. Proposed biochemical pathway for the metabolism of rumeleniccis-

F. Destaillats et al. / J. Chromatogr. B 820 (2005) 15–22 17

Fig. 2. Mass spectra of DMOX derivatives ofcis-8,cis-11,trans-13,cis-17 20:4 (A),cis-5,cis-8,cis-11,trans-13,cis-17 20:5 (C),cis-7,cis-10,cis-13,trans-15,cis-19 22:5 (E),cis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 (G) acids, and methyl esters derivatives ofcis-8,cis-11,trans-13,cis-17 20:4 (B),cis-5,cis-8,cis-11,trans-13,cis-17 20:5 (D),cis-7,cis-10,cis-13,trans-15,cis-19 22:5 (F),cis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 (H) acids. Fragmentations occurredtowards the carboxyl group.

of its methyl ester derivative, shown inFig. 2B, exhibits anintense fragment atm/z= 249 which corresponds to a loss ofa 69 amu (C5H9) fragment from the parental molecular ionatm/z= 318.

The DMOX derivative spectrum of thecis-5,cis-8,cis-11,trans-13,cis-17 20:5 acid is shown inFig. 2C. The molecu-lar ion atm/z= 355 confirms the eicosapentaenoic acid struc-ture. The intense ion fragment atm/z= 286 confirmed a bis-methylene interrupted structure, and allow the location of

the�13 and�17 double bonds. Ion fragments atm/z= 180,192, 220 and 232 allow the location of the double bonds inpositions�8 and�11, respectively. The odd numbered ionfragment atm/z= 153 is characteristic for the�5 ethylenicdouble bond and resulted from the ionization of the nitro-gen atom followed by cyclization and cleavage of the restof the aliphatic chain[3]. The corresponding methyl esterderivative spectrum is presented inFig. 2D. Similarly tothe DMOX derivative of 20:4 acid, the methyl ester deriva-


Fig. 2. (Continued)

tive spectrum presents a characteristic ion fragment at 247corresponding to M+ − 69 amu. Moreover, the spectrum ex-hibits an intense ion fragment atm/z= 180 corresponding toa cleavage between carbons 10 and 11. Indeed, this diagnos-tic ion is also present in the DMOX spectrum (Fig. 2C) atm/z= 220.

Charge remote fragmentation mechanism similar to thatproposed by Dobson et al.[16], leads to the formationof ion fragment at M+ − 69 amu for both DMOX andmethyl ester metabolites of 9,11,15 18:3 acid is givenin Fig. 3 [17]. The abstraction of a bis-allylic hydro-

gen radical consecutively to the formation of a positiveradical molecular ion, results in a series of delocaliza-tion that gives, after disruption of the C15 C16 sigmabond, a stable penta-unsaturated ion fragment with fourconjugated ethylenic double bonds. Formation of intenseion fragments at M+ − 135 and M+ − 136 amu for DMOXand methyl ester derivatives, respectively, could be theresult of allylic cleavage of then-12 ethylenic doublebond.

The DMOX derivative spectrum ofcis-7,cis-10,cis-13,trans-15,cis-19 22:5 acid is given inFig. 2E. The in-


Fig. 2. (Continued)

tense ion fragment atm/z= 383 and the ion fragmentat m/z= 368, corresponding to a loss of the terminalmethyl group, confirmed the docosapentaenoic acid struc-ture. The intense ion fragment atm/z= 314 correspond-ing to the cleavage at the center of the bis-methylene in-terrupted double bond allow the location of both�19and �15 double bonds. Additional intense allylic frag-ment at m/z= 248 corroborated the location of�13,15conjugated system. Ion fragments atm/z= 220, 208 and180, 168 allow the location of double bonds�10 and

�7, respectively. The key diagnostic ion for the DMOXderivative of 7,10,13,15,19 22:5 are the intense ion frag-ments atm/z= 314 and 248 representing the loss of 69and 135 amu, respectively. The methyl ester spectrumof this FA (Fig. 2F) exhibits similar ion fragmentationand presents two intense ion fragments atm/z= 275 and208.

The DMOX derivative spectrum of the 4,7,10,13,15,1922:5 acid which is an intermediate (Fig. 1) in the forma-tion of the partially conjugated docosahexaenoic acid, is il-


Fig. 2. (Continued).

lustrated inFig. 2G. The molecular ion atm/z= 381 andthe ion fragment atm/z= 366 corresponding to the loss ofthe terminal methyl group confirmed the docosahexanoicacid structure. The intense ion fragment atm/z= 312 con-firmed the bis-methylene interrupted structure and corrobo-rates both location of�19 and�15 ethylenic double bonds.The ion fragment atm/z= 246 confirmed the�13,15 con-jugated system. Methylene interrupted�7 and�10 dou-ble bonds can be assignated by ion fragments atm/z= 166,178 andm/z= 206 and 232, respectively. Moreover, ion frag-ment atm/z= 220 is indicative of the loss of vinylic car-

bon at position 11 (loss of 12 amu fromm/z= 232). Sim-ilarly, the loss of vinylic carbon at position 16 (loss of12 amu) resulted in ion fragment atm/z= 286, from 298.The odd numbered ion fragment atm/z= 139 is an unam-biguous evidence for the location of a�4 double bond[3].The methyl ester derivative spectrum of this PUFA is givenin Fig. 2H, and the observed characteristic ion fragments atM+ − 69 (m/z= 273) and M+ − 136 (206), are also present inthis spectrum.

The methyl ester derivatives of the metabolites ofcis-9,trans-11,cis-15 18:3 (rumelenic) acid exhibit spe-


Fig. 3. Proposed chemical mechanism for the formation of ion fragment atm/z= M+ − 69 by charge remote fragmentation in electronic impact ioniza-tion mass spectrometry ofcis-5,cis-8,cis-11,trans-13,cis-17 20:5 acid as itsDMOX and methyl ester derivatives.

cific mass spectral properties: intense ion fragments atm/z= M+ − 69 corresponding to the cleavage at the cen-ter of the bis-methylene interrupted double bond systemare observed in all cases. Moreover, mass spectra of theC20:5, C22:5, C22:6 methyl ester derivatives of this par-tially conjugated PUFA group, exhibit an intense ion frag-ment atm/z= M+ − 136, specific for the allylic cleavageof the n-12 ethylenic double bond. Indeed, these ion frag-ments, not usually observed for other PUFA methyl es-ter derivatives, permit their accurate detection in GC–MS(Fig. 4). Our mass spectral observations are correlated withthe analysis of their respective DMOX derivatives spec-tra.

Fig. 4. Reconstructed ion chromatograms for methyl esters derivativesof cis-7,cis-10,cis-13,trans-15,cis-19 22:5 (1) andcis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 (2) acids isolated from rat liver lipid (total ion currentB, TIC B). TIC A corresponds to a liver lipid sample from rat non-fed withrumelenic acid.

4. Conclusion

The DMOX derivatization, widely used for lipid analysis,requires high reaction temperature (150–190◦C) and longreaction time (6–12 h) that may result in partial degradationof PUFA [1]. We have shown that methyl esters derivatives,used for routine fatty acid profile quantification by GC–FID,may be used for structural authentification of metabolites ofrumelenic acid by GC–MS.

Acknowledgements

The authors wish to express their gratitude to Dr. L. Bretil-lon and Dr. J.M. Chardigny for helpful discussions and re-vising the manuscript, and S. Gregoire and S. Almanza fortechnical assistance. We acknowledge financial support ofNatural Sciences and Engineering Council of Canada, and ofFonds FCAR (Gouvernement du Quebec). The authors arealso grateful to Fondation de l’Universite Laval for a Ph.D.Scholarship to F. Destaillats.

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